Farley et al. 2024

Trifunctional Sphinganine: A New Tool to Dissect Sphingolipid Function

Study details

Authors

Scotland E. Farley, Frank Stein, Per Haberkant, Fikadu G. Tafesse, Carsten Schultz

Journal

ACS Chemical Biology

https://doi.org/10.1021/acschembio.3c00554

Abstract

Functions of the sphingolipids sphingosine and sphinganine in cells are not well established. While some signaling roles for sphingosine have been elucidated, the closely related sphinganine has been described only insofar as it does not elicit many of the same signaling responses. The underlying mechanisms behind the cell biological differences between these lipids are not well understood. Here, we prepared multifunctionalized derivatives of the two lipid species that only differ in a single double bond of the carbon backbone. Using these novel probes, we were able to define their spatiotemporal distribution within cells. Furthermore, we used these tools to systematically map the protein interactomes of both lipids. The lipid-protein conjugates, prepared through photo-crosslinking in live cells and extraction via click chemistry to azide beads, revealed significant differences in the captured proteins, highlighting their distinct roles in various cellular processes. This work elucidates mechanistic differences between these critical lipids and sets the foundation for further studies on the functions of sphingosine and sphinganine.

Lipid probes utilized

Trifunctional Sphingosine

Trifunctional Sphinganine

Cell line analyzed

Huh7

Uncaging & Crosslinking timeline

Lipid Probe	Uptake time	Uncaging time	Interaction time	Crosslinking time
Sph	60 min	5 min	15 min	5 min
Spa	60 min	5 min	15 min	5 min

Mass spectrometry quantification method

16-channel Tandem Mass Tagging (TMT16)

Additional sample preparation ?

Data wrangling

Use the download button below to download the R script used to wrangle the authors’ original submission into the data visualization tools on the Lipid Interactome:

Use the download button below to download the original data file prior to wrangling:

Data visualization

Volcano plots

Volcano plots depict the relative enrichment of each protein versus control (x-axis) and the statistical significance of each protein following a Student’s t-test (y-axis, -log10 transformed). The left panel depicts the results of a pulldown using trifunctionalized sphingosine; the right panel depicts the results of the pulldown using trifunctionalized sphinganine. Black proteins are unenriched or depleted in the presence of probe, Purple proteins are enriched to the probe but fall below a FDR or p-value threshold of significance, and Orange proteins surpass the threshold of significance (need details of threshold).

Ranked-order plots

Ranked-order plots depict the relative enrichment of each protein versus control (y-axis) from lowest to highest. The left panel depicts the results of a pulldown using trifunctionalized sphingosine; the right panel depicts the results of the pulldown using trifunctionalized sphinganine. Black proteins are unenriched or depleted in the presence of probe, Purple proteins are enriched to the probe but fall below a FDR or p-value threshold of significance, and Orange proteins surpass the threshold of significance (need details of threshold).

MA Plots

MA plots depict the average abundance of each protein in the crosslinked and uncrosslinked conditions (x-axis) versus the log2-transformed fold-change between the crosslinked and uncrosslinked conditions (y-axis). The left panel depicts the results of a pulldown using trifunctionalized sphingosine; the right panel depicts the results of the pulldown using trifunctionalized sphinganine. Black proteins are unenriched or depleted in the presence of probe, Purple enriched candidates are defined as proteins with a false discovery rate less than 0.2 and a fold change of at least 1.5-fold, and Orange enriched hits are defined as proteins with a false discovery rate less than 0.05 and a fold change of at least 2-fold in the +UV over the -UV).

Gene Ontology Analysis

In beta: GO analysis still under development

Cell Compartment

Molecular Function

Biological Process

(No BP pathways identified among enriched hits and candidates.)

Figure 1: GO Dot plots display the enrichment of GO terms among the proteins enriched to the probe. For these analyses, only proteins categorized as “enriched candidates” and “enriched hits” were subject to GO analysis. The Cell Compartment analysis assesses whether the list of enriched proteins contains a statistically significant number of proteins in the same cellular region; appropriately, the Molecular Function and Biological Process analyses does the same for molecular function and biological process, respectively. Click here for more information about Gene Ontology analysis.

Data exploration

Check the boxes below to filter the dataset by which lipid probe was used in the pulldown and by significance thresholds.

Lipid probe in pulldown:

Sph

Spa

Enrichment criteria:

enriched hit

enriched candidate

no hit

This datatable contains all the data plotted above. Check the boxes below to filter the dataset by which lipid probe was used in the pulldown and by significance thresholds. ‘No hit’ proteins are unenriched or depleted in the presence of probe, ‘enriched candidates’ are defined as proteins with a false discovery rate less than 0.2 and a fold change of at least 1.5-fold, and ‘enriched hits’ are defined as proteins with a false discovery rate less than 0.05 and a fold change of at least 2-fold in the +UV over the -UV.

Data download